Research has produced many peptides that have potential as therapeutic compositions. Yet realizing and exploiting the full therapeutic potential of peptides directed against intracellular targets has yet to be achieved, for a variety of reasons. One of the most important of these is that most therapeutic peptides do not possess the ability to cross cell membranes to reach their therapeutic targets. One solution to this problem is the use of carrier peptides that act to ferry a cargo peptide into a target cell.
There are a number of notable examples of carrier peptides which are effective to facilitate the crossing of a target cell's membrane by a cargo peptide. One example is a peptide sequence derived from the TAT protein of the HIV virus. See U.S. Pat. No. 6,316,003, which is hereby incorporated by reference in its entirety. Another well known carrier peptide sequence is the “poly-Arg” sequence. See, e.g., U.S. Pat. No. 6,306,993.
In many cases, the use of a disulfide bond to link the carrier and cargo peptides, producing the therapeutic peptide construct, is an effective strategy to solve the problem of targeting soluble peptides to intracellular targets. One theory explaining the usefulness of disulfide bonds holds that once the carrier-cargo construct enters a target cell, the two peptides can separate through disulfide bond reduction. This separation in the intracellular environment may allow a greater diffusion of cargo peptides within the cell as opposed to other linkage mechanisms which maintain the carrier-cargo link. With this said, however, the administration of therapeutic peptides still suffers from numerous challenges, such as disulfide bond exchange, proteolytic degradation and efficiency of cellular uptake. Methods directed to controlling these issues will increase the stability and potency of therapeutic peptides.
One way to increase the potency of a therapeutic peptide comprising a carrier peptide disulfide bonded to a cargo peptide is to reduce disulfide bond exchange. Disulfide bond exchange reduces the amount of a carrier-cargo peptide construct in a given sample by allowing a carrier peptide to exchange its cargo peptide for another carrier peptide, thus resulting in a carrier-carrier construct and a cargo-cargo construct. The carrier-only construct will have no therapeutic effect. The cargo-cargo construct will have a tremendously reduced, if not completely eliminated effect, since the carrier peptide enables the delivery of the cargo to its intracellular target. As such, the problem of controlling disulfide bond exchange is important to maximizing the therapeutic potential of a carrier-cargo peptide construct.
Another problem facing the use of therapeutic peptides is proteolytic degradation. Peptides are notoriously unstable molecules and frequently labile to proteolytic attack when administered to a subject. Labile carrier peptides which degrade upon administration will reduce or even eliminate the efficacy of the cargo peptide because the cargo depends upon the carrier peptide to reach the intracellular target. Thus, methods to control or eliminate the labile nature of therapeutic peptides are also important to maximizing a carrier-cargo peptide's therapeutic potential.
Increasing the efficiency of cellular uptake of a therapeutic peptide is yet another problem which can reduce the efficacy or potency of a therapeutic peptide. Optimization of carrier peptide sequences and placement relative to the cargo peptide provide methods for increasing the stability and potency of therapeutic peptide constructs.